Immunotherapeutic stress protein-peptide complexes against cancer

ABSTRACT

Disclosed is a method for inhibiting the proliferation of a tumor in a mammal. The method involves the steps of (a) isolating a stress protein-peptide complex from tumor cells previously removed from the mammal and (b) administering the isolated stress protein-peptide complex back to the mammal in order to stimulate in the mammal an immune response against the tumor from which the complex was isolated. Stress protein-peptide complexes having particular utility in the practice of the instant invention include the Hsp70-peptide, Hsp90-peptide and gp96-peptide complexes.

FIELD OF THE INVENTION

[0001] The application relates generally to the field of cancer therapy,in particular, to the immunotherapy of human cancer.

BACKGROUND OF THE INVENTION

[0002] It has been found that inbred mice and rats can be immunizedphrophylactically against tumors derived from mice and rats of the samegenetic background (Gross (1943) Cancer Res. 3:323-326; Prehn et al.(1957) J. Natl. Cancer Inst. 18:769-778; Klein et al. (1960) Cancer Res.20:1561-1572; Old et al. (1962) Ann NY Acad. Sci. 101:80-106; forreview, see Srivastava et al. (1988) Immunology Today 9:78-83). Thesestudies not only showed that mice vaccinated with inactivated cancercells become immunized against subsequent challenges of live cancercells but also demonstrated the existence of tumor-specific antigens.

[0003] Further studies revealed that the phenomenon of prophylacticallyinduced immunity is tumor-specific. Although mice can be specificallyimmunized against the tumor cells that were used to immunize them theystill remain sensitive to challenges with other unrelated tumors(Basombrio (1970) Cancer Res. 30:2458-2462, Globerson et al. (1964) J.Natl. Cancer Inst. 32:1229-1243). The demonstration of immunogenicity ofcancer cells led to a search for the cancer-derived molecules whichelicit resistance to tumor challenges. The general approach was tofractionate cancer cell-derived proteins and test them individually fortheir ability to immunize mice against the cancers from which thefractions were prepared (see Srivastava et al. (1988) supra; Old (1981)Cancer Res. 41:361-375). A number of proteins have been identified bythis method, however, a large proportion of these proteins are relatedto a class of proteins known as stress-induced proteins or stressproteins (Lindquist et al. (1988) Annual Rev. Genet. 22:631-677).Because the stress proteins are among the most highly conserved andabundant proteins in nature, they are unlikely candidates for tumorspecific antigens. Stress proteins have subsequently been shown to noncovalently associate with a variety of peptides thereby to form stressprotein-peptide complexes (Gething et al. (1992) Nature 355:33-45;Lindquist et al. (1988) supra; Young (1990) Annu. Rev. Immunol.8:401-420; Flynn et al. 1991) Nature 353:726-730).

[0004] Studies have also shown that stress protein-peptide complexeslose their immunogenicity upon treatment with ATP (Udono et al. (1993)J. Exp. Med. 178:1391-1396). This treatment is known to dissociate thestress-protein peptide complex into its stress protein and peptidecomponents. Considering that there are no differences in the structureof stress proteins derived from normal and tumor cells, and that stressproteins bind a wide spectrum of peptides in an ATP dependent manner itappears that the antigenicity of the stress protein-peptide complexresults not from the stress protein per se, but from the peptideassociated with the stress protein.

[0005] One of the major conceptual difficulties in cancer immunotherapyhas been the possibility that human cancers, like cancers ofexperimental animals, are antigenically distinct. Clearly, there is somerecent evidence for existence of common tumor antigens (Kawakami et al.(1992) J. Immunol. 148:638-643; Darrow et al. (1989) J. Immunol.142:3329-3334), and this augurs well for prospects of cancerimmunotherapy. Nonetheless, in light of the overwhelming evidence fromexperimental and human systems, it is reasonable to assume that at thevery least, human tumors would show tremendous antigenic diversity andheterogeneity.

[0006] The prospect of identification of the immunogenic antigens ofindividual tumors from cancer patients (or even of ‘only’ severaldifferent types of immunogenic antigens in case the antigens areshared), is daunting to the extent of being impractical. Conventionalcancer therapies typically are based on the isolation andcharacterization of specific antigenic determinants which then maybecome the target for subsequent immunotherapies. In addition, althoughstudies have demonstrated that mammals can be immunized prophylacticallyagainst tumors derived from mammals of the same genetic background,heretofore it has not been appreciated that a mammal harboring a tumorcan be therapeutically immunized with a composition derived from its owntumor as a means of treating a cancer preexisting in the mammal.

[0007] Accordingly, it is an object of the instant invention to providea novel method for therapeutically inhibiting proliferation of tumors ina mammal. The method described herein does not require the isolation andcharacterization of specific antigenic determinants, and accordinglyprovides a more rapid approach for making and using immunogeniccompositions effective in inhibiting the proliferation of specificpredetermined tumors in mammals.

[0008] This and other objects and features of the invention will beapparent from the description and claims which follow.

SUMMARY OF THE INVENTION

[0009] The observation that stress proteins chaperone the antigenicpeptides of the cells from which they are derived provides an approachfor readily isolating antigenic peptides for a preselected tumor. Onceisolated, the stress protein-peptide complexes are administered back tothe animal from which they were derived in order to elicit an immuneresponse against a preexisting tumor. Accordingly, this approachcircumvents the necessity of isolating and characterizing specific tumorantigens and enables the artisan to readily prepare immunogeniccompositions effective against a preselected tumor.

[0010] In its broadest aspect, the invention provides a method forinhibiting proliferation of a preselected tumor in a mammal. The methodcomprises administering to the mammal undergoing therapy a compositioncomprising a pharmaceutically acceptable carrier in combination with astress protein-peptide complex. The complex having been isolated from atumor cell previously excised from the mammal and characterized in thatit is operative to initiate in the mammal an immune response against thetumor cells from which it was derived. The complex subsequently isadministered back to the mammal in an amount sufficient to elicit in themammal an immune response against the tumor cells thereby to inhibitproliferation of any tumor cells still remaining in the mammal.

[0011] It is contemplated that this approach may be used in combinationwith other conventional cancer therapies which include, for example,surgery, radiation therapy and chemotherapy. For example, followingsurgical excision of cancerous tissue the artisan, using the principlesdescribed herein, may isolate stress protein-peptide complexes from theexcised tissue and administer the complex back to the mammal. Thecomplex subsequently induces a specific immune response against anyremaining tumor cells that were not excised during surgery. The approachis amenable to cancer therapy when the primary tumor has metastasized todifferent locations with the body.

[0012] The term “tumor” as used herein, is understood to mean anyabnormal or uncontrolled growth of cells which may result in theinvasion of normal tissues. It is contemplated also that the termembraces abnormal or uncontrolled cell growths that have metastasized,i.e., abnormal cells that have spread from a primary location in thebody (i.e., primary tumor) to a secondary location spatially removedfrom the primary tumor.

[0013] The term “stress protein” as used herein, is understood to meanany cellular protein which satisfies the following criteria. It is aprotein whose intracellular concentration increases when a cell isexposed to stressful stimuli, is capable of binding other proteins orpeptides, and is capable of releasing the bound proteins or peptides inthe presence of adenosine triphosphate (ATP) and/or low pH. Stressfulstimuli include, but are not limited to, heat shock, nutrientdeprivation, metabolic disruption, oxygen radicals, and infection withintracellular pathogens.

[0014] The first stress proteins to be identified were the heat shockproteins (Hsp's). As their name suggests, Hsp's typically are induced bya cell in response to heat shock. Three major families of mammalianHsp's have been identified to date and include Hsp60, Hsp70 and Hsp90.The numbers reflect the approximate molecular weight of the stressproteins in kilodaltons. The members of each of the families are highlyconserved, see for example, Bardwell et al. (1984) Proc. Natl. Acad.Sci. 81:848-852; Hickey et al. (1989) Mol. Cell Biol. 9:2615-2626;Jindal (1989) Mol. Cell. Biol. 9:2279-2283, the disclosures of which areincorporated herein by reference. Members of the mammalian Hsp90 familyidentified to date include cytosolic Hsp90 (also known as Hsp83) and theendoplasmic reticulum counterparts Hsp90 (also known as Hsp83), Hsp87,Grp94 (also known as ERp99) and gp96. See for example, Gething et al.(1992) Nature 355:33-45 the disclosure of which is incorporated hereinby reference. Members of the Hsp70 family identified to date include:cytosolic Hsp70 (also known as p73) and Hsc70 (also known as p72); theendoplasmic reticulum counterpart BiP (also known as Grp78); and themitochondrial counterpart Hsp 70 (also known as Grp75), Gething et al.(1992) supra. To date, members of the mammalian Hsp60 family have onlybeen identified in the mitochondria, Gething et al. (1992) supra.

[0015] In addition, it has been discovered that the Hsp-60, Hsp-70 andHsp-90 families are composed of proteins related to the stress proteinsin amino acid sequence, for example, having greater than 35% amino acididentity, but whose expression levels are not altered by stressfulstimuli. Accordingly, it is contemplated that the definition of stressprotein, as used herein, embraces other proteins, muteins, analogs, andvariants thereof having at least 35% to 55%, preferably 55% to 75%, andmost preferably 75% to 85% amino acid identity with members of the threefamilies whose expression levels in a cell are stimulated in response tostressful stimuli.

[0016] The term “peptide”, as used herein, is understood to mean anyamino acid sequence isolated from a mammalian tumor cell in the form ofa stress protein-peptide complex.

[0017] The term “immunogenic stress protein-peptide complex”, as usedherein, is understood to mean any complex which can be isolated from amammalian tumor cell and comprises a stress protein non covalentlyassociated with a peptide. The complex is further characterized in thatit is operative to induce in the mammal an immune response against thetumor cells from which the complex was derived.

[0018] The term “immune response” is understood to mean any cellularprocess that is produced in the mammal following stimulation with anantigen and is directed toward the elimination of the antigen from themammal. The immune response typically is mediated by one or morepopulations of cells characterized as being lymphocytic and/orphagocytic in nature.

[0019] In a more specific aspect of the invention, the stress protein inthe stress protein-peptide complex is selected from the group consistingof Hsp70, Hsp90 and gp96. Stress protein-peptide complexes which includeHsp70-peptide, Hsp90-peptide and gp96-peptide complexes may be isolatedsimultaneously from a batch of tumor cells excised from a mammal. Duringimmunotherapy it is contemplated that one or more of the aforementionedcomplexes may be administered to the mammal in order to stimulate theoptimal immune response against the tumor.

[0020] It is contemplated that the method described herein isparticularly useful in the treatment of human cancer. However, it iscontemplated that the methods described herein likewise will be usefulin immunotherapy of cancers in other mammals, for example, farm animals(i.e., cattle, horses, goats, sheep and pigs) and household pets (i.e.,cats and dogs).

[0021] In another aspect of the invention, it is contemplated that theimmune response is effected by means of a T cell cascade, and morespecifically by means of a cytotoxic T cell cascade. The term “cytotoxicT cell”, as used herein, is understood to mean any T lymphocyteexpressing the cell surface glycoprotein marker CD8 that is capable oftargeting and lysing a target cell which bears a class Ihistocompatibility complex on its cell surface and is infected with anintracellular pathogen.

[0022] In another aspect of the invention, the stress protein-peptidecomplexes may be administered to the mammal in combination with atherapeutically active amount of a cytokine. As used herein, the term“cytokine” is meant to mean any secreted polypeptide that influences thefunction of other cells mediating an immune response. Accordingly, it iscontemplated that the complex can be coadministered with a cytokine toenhance the immune response directed against the tumor. Preferredcytokines include, but are not limited to, interleukin-1α (IL-1α),interleukin-1β (IL-1β), 1β), interleukin-2 (IL-2), interleukin-3 (IL-3),interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9),interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12),interferon α (IFNα), interferon β (IFNβ), interferon γ (IFNγ), tumornecrosis factor α (TNF∝), tumor necrosis factor β (TNFβ), granulocytecolony stimulating factor (G-CSF), granulocyte/macrophage colonystimulating factor (GM-CSF), and transforming growth factor β (TGF-β).

[0023] The complex may be administered to a mammal when combined with aconventional pharmaceutically acceptable carrier, adjuvant, or excipientusing techniques well known in the art. The dosage and means ofadministration of the family of stress protein-peptide complexesnecessarily will depend upon a variety of factors such as the stabilityof the complex under physiological conditions, the effectiveness of thecomplex at eliciting an immune response, the size and distribution ofthe tumor, and the age, sex and weight of the mammal undergoing therapy.

[0024] Typically, the complex should be administered in an amountsufficient to initiate in the mammal an immune response against thetumor from which the complex was derived and in an amount sufficient toinhibit proliferation of the tumor cells in the mammal. The amount ofstress protein-peptide complex administered preferably is in the rangeof about 1-1000 micrograms of complex/kg body weight of themammal/administration, and most preferably about 100-250 micrograms ofcomplex/kg body weight of the mammal/administration. It is contemplatedthat typical dose will be in the range of about 5 to about 20 mg for ahuman subject weighing about 75 kg. In addition, it is contemplated thatthe strength of the immune response may be enhanced by repeatedlyadministering the complex to the individual. The mammal preferablyreceives at least two doses of the stress protein-peptide complex atweekly intervals. If necessary, the immune response may be boosted at alater date by subsequent administration of the complex. It iscontemplated, however, that the optimal dosage and immunization regimenmay be found by routine experimentation by one skilled in the art.

DETAILED DESCRIPTION

[0025] The invention is based on the observation that stressprotein-peptide complexes chaperone antigenic peptides of the cells fromwhich they are derived. Conventional cancer therapies are based upon theisolation an characterization of tumor specific antigens which thenbecome the target for a specific therapeutic regime. Because of theantigenic diversity of mammalian-cancers the isolation andcharacterization of specific tumor antigens for each specific tumor canbe impractical. The instant invention thus provides an alternativeapproach to cancer immunotherapy by obviating the necessity of isolatingand characterizing tumor specific antigens for each tumor being treated.

[0026] The invention described herein provides a method for inhibitingproliferation of a preselected tumor in a mammal. The method comprisesisolating or obtaining tumor cells from the mammal undergoing therapy.This is accomplished readily using conventional surgical procedures wellknown in the art. Typically, tumor cells are excised from the mammalduring routine surgical recision of the tumor. The method then involvesisolating stress protein-peptide complexes from the excised tumor cells.This is accomplished using any one of the isolation procedures describedin detail herein below. The stress protein-peptide complexes arecharacterized in that when they are administered back to the mammal theyare capable of initiating a specific immune response against the sametype of tumor cells that they were derived from. Finally, the methodcomprises the step of administering back to the mammal the isolatedstress protein-peptide complex in an amount sufficient to elicit in themammal an immune response against the tumor cells thereby inhibitingproliferation of any tumor cells remaining in the mammal.

[0027] It is contemplated that this approach may be used in combinationwith one or more conventional cancer therapies which include, forexample, surgery, radiation therapy and chemotherapy. For example,following surgical excision of cancerous tissue the artisan, using theprinciples described herein, may isolate stress protein-peptidecomplexes from the excised tissue and administer the complex back to themammal. The complex then induces in the mammal a specific immuneresponse against any tumor cells that were not removed during surgery.Alternatively, the method described herein provides a novel approach fortreating cancer when the primary tumor has metastasized to multiplelocations with the body. For example, when the cancer has metastasized,making surgical intervention impractical, a stress protein-peptidecomplex may be used either alone or in combination with another standardchemotherapeutic agent in the treatment of the cancer.

[0028] It is contemplated that the invention has particular utility inthe immunotherapy of human cancer, however, it is appreciated that themethodologies described herein may be applied to the treatment ofcancers occurring in, for example, farm animals (i.e., cattle, horses,sheep, goats and pigs) and household pets (i.e., cats and dogs).

[0029] The main advantage this approach has over conventionalmethodologies is that it is not necessary to isolate and characterizethe tumor specific antigen for each tumor. Once the stressprotein-peptide complex has been isolated it is simply administered backthe mammal without further characterization. Since the procedures forisolating the immunogenic complexes are routine and well known in theart, the artisan may rapidly and routinely prepare a specificimmunogenic composition “tailor-made” for each individual being treated.

[0030] Another advantage of the instant method over previousmethodologies is that the administration of purified stressprotein-peptide complexes back to the individual from which they werederived eliminates the risk of inoculating the mammal undergoing therapywith potentially transforming agents (i.e., transforming DNA) and/orimmunosuppressive agents which can be an issue when the complex ispresent in a biochemically undefined tumor or tumor extract. Inaddition, stress protein-peptide complexes can induce significant tumorimmunity in the absence of adjuvants. Accordingly, while adjuvants mayfurther enhance the immunotherapeutic properties of the complex, theiravailability is not a pre-condition for inducing a significant immuneresponse.

[0031] It is contemplated that this method can be used in the treatmentof a variety of tumors, for example, tumors that are mesenchymal inorigin (sarcomas) i.e., fibrosarcomas; myxosarcomas; liposarcomas;chondrosarcomas; osteogenic sarcomas; angiosarcomas; endotheliosarcomas;lymphangiosarcomas; synoviosarcomas; mesotheliosarcomas; Ewing's tumors;myelogenous leukemias; monocytic leukemias; malignant lymphomas;lymphocytic leukemias; plasmacytomas; leiomyosarcomas andrhabdomyosarcoma.

[0032] In addition, it is contemplated that this method can be used inthe treatment of tumors that are epithelial in origin (carcinomas) i.e.,squamous cell or epidermal carcinomas; basal cell carcinomas; sweatgland carcinomas; sebaceous gland carcinomas; adenocarcinomas; papillarycarcinomas; papillary adenocarcinomas; cystadenocarcinomas; medullarycarcinomas; undifferentiated carcinomas (simplex carcinomas);bronchogenic carcinomas; bronchial carcinomas; melanocarcinomas; renalcell carcinomas; hepatocellular carcinomas; bile duct carcinomas;papillary carcinomas; transitional cell carcinomas; squamous cellcarcinomas; choriocarcinomas; seminomas; embryonal carcinomas malignantteratomas and teratocarcinomas. Generic methodologies useful in thepreparation of compositions effective at inducing an immune responseagainst these tumors are discussed in detail herein below.

[0033] Although not wishing to be bound by theory, it is contemplatedthat the stress protein-peptide complexes stimulate an immune responseagainst the tumor cells from which they are derived by means of a T cellcascade. Previous experiments have demonstrated that mice immunizedprophylactically with stress protein-peptide preparations derived from atumor originating in the same strain of mouse or rat developimmunological resistance to the tumor from which it was isolated. Themice, however, fail to develop immunity against antigenically distincttumors. Furthermore, stress protein-peptide complexes derived fromnormal tissues do not elicit resistance to any tumors tested. See forexample, Srivastava et al. (1984) Int. J. Cancer 33:417; Srivastava etal. (1986) Proc. Natl. Acad. Sci. USA 83:3407; Palladino et al. (1987)Cancer Res. 47:5074; Feldweg et al. (1993) J. Cell Biochem. Suppl.17D:108 (Abst.); Udono et al. (1993) J. Cell. Biochem. Suppl. 17D:113and Udono (1993) J. Exp. Med.178:1391-1396, the disclosures of which areincorporated herein by reference. Recently, it has been establishedprophylactic immunity typically is mediated by means of a T cellcascade, more specifically by means of a cytotoxic T cell cascade. Seefor example, Blachere et al. (1993) J. Immunother. 14:352-356, thedisclosure of which is incorporated by reference herein. Accordingly, itis contemplated that the stress-protein complexes may also mediate theireffect therapeutically by a similar mechanism; specifically, via acytotoxic T cell cascade.

[0034] It is contemplated that the stress protein-peptide complexestypically will be isolated directly from tumor tissue excised from themammal being treated. Under certain conditions, however, the amount oftumor tissue available for isolation of the complex may be limiting.Accordingly, it is contemplated that the excised tumor tissue may beproliferated using techniques well known in the art prior to theisolation of the stress protein-peptide complexes. For example, theexcised tumor tissue may be proliferated either in vivo, for example, bytransfecting a nude mouse with a sample of the tumor tissue, or invitro, for example, by serially passaging the tumor cells in culture.The proliferated tumor tissue subsequently can be harvested and used asa starting material for the isolation of the stress protein-peptidecomplex.

[0035] Stress proteins useful in the practice of the instant inventionmay be defined as any cellular protein that satisfies the followingcriteria. It is a protein whose intracellular concentration increaseswhen a cell is exposed to a stressful stimuli, is capable of bindingother proteins or peptides, and is capable of releasing the boundproteins or peptides in the presence of adenosine triphosphate (ATP) orlow pH.

[0036] The first stress proteins to be identified were the Hsp's whichare synthesized in a cell in response to heat shock. To date, threemajor families of mammalian Hsp's have been identified and includeHsp60, Hsp70 and Hsp90 where the numbers reflect the approximatemolecular weight of the stress proteins in kilodaltons. Many members ofthese families were found subsequently to be induced in response toother stressful stimuli including, but not limited to, nutrientdeprivation, metabolic disruption, oxygen radicals, and infection withintracellular pathogens. See for example: Welch (May 1993) ScientificAmerican 56-64; Young (1990) supra; Craig (1993) Science 260:1902-1903;Gething et al (1992) supra; and Lindquist et al. (1988) supra, thedisclosures of which are incorporated herein by reference. It iscontemplated that mammalian stress proteins belonging to all threefamilies may be useful in the practice of the instant invention.

[0037] The major stress proteins accumulate to very high levels instressed cells but occur at low to moderate levels in cells that havenot been stressed. For example, the highly inducible mammalian Hsp70 ishardly detectable at normal temperatures but becomes one of the mostactively synthesized proteins in the cell upon heat shock (Welch et al.(1985), J. Cell. Biol. 101:1198-1211). In contrast, Hsp90 and Hsp60proteins are abundant at normal temperatures in most, but not all,mammalian cells and are further induced by heat (Lai et al. (1984), Mol.Cell. Biol. 4:2802-10; van Bergen en Henegouwen et al. (1987), GenesDev., 1:525-31).

[0038] Members of the mammalian Hsp90 family identified to date includecytosolic Hsp90 (also known as Hsp83).and the endoplasmic reticulumcounterparts Hsp90 (also known as Hsp83), Hsp87, Grp94 (also known asERp99) and gp96 (Gething et al. (1992) supra). Members of the Hsp70family identified to date include: cytosolic Hsp70 (also known as p73)and Hsc70 (also known as p72), the endoplasmic reticulum counterpart BiP(also known as Grp78) and the mitochondrial counterpart Hsp 70 (alsoknown as Grp75), Gething et al. (1992) supra. To date, members of themammalian Hsp60 family have only been identified in the mitochondria,Gething et al. (1992) supra.

[0039] Stress proteins are among the most highly conserved proteins inexistence. For example, DnaK, the Hsp70 from E. coli has about 50% aminoacid sequence identity with Hsp70 proteins from eukaryotes (Bardwell etal. (1984) Proc. Natl. Acad. Sci. 81:848-852). The Hsp60 and Hsp90families similarly exhibit high levels of intrafamilial conservation(Hickey et al. (1989) Mol. Cell Biol. 9:2615-2626; Jindal (1989) Mol.Cell. Biol. 9:2279-2283). In addition, it has been discovered that theHsp60, Hsp70 and Hsp90 families are composed of proteins that arerelated to the stress proteins in sequence, for example, having greaterthan 35% amino acid identity, but whose expression levels are notaltered by stress. Therefore it is contemplated that the definition ofstress protein, as used herein, embraces other proteins, muteins,analogs, and variants thereof having at least 35% to 55%, preferably 55%to 75%, and most preferably 75% to 85% amino acid identity with membersof the three families whose expression levels in a cell are enhanced inresponse to a stressful stimulus.

[0040] The immunogenic stress protein-peptide complexes of the inventionmay include any complex containing a stress protein non covalentlyassociated with a peptide that is capable of inducing an immune responsein a mammal. Preferred complexes include, but are not limited to,Hsp70-peptide, Hsp90-peptide and gp96- peptide complexes. For example,the mammalian stress protein gp96 which is the endoplasmic reticulumcounterpart of the cytosolic Hsp90 may be used in the practice of theinstant invention.

[0041] Typical procedures for isolating stress protein-peptide complexesuseful in the practice of the instant invention are set forth in detailbelow.

Purification of Hsp70-peptide Complexes

[0042] The purification of Hsp70-peptide complexes has been describedpreviously, see for example, Udono et al. (1993) supra.

[0043] Initially, tumor cells are suspended in 3 volumes of 1X Lysisbuffer consisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2mM CaCl_(2,) 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF).Then, the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 min and then homogenized in a douncehomogenizer until >95% cells are lysed.

[0044] Then the lysate is centrifuged at 1000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith phosphate buffered saline (PBS) containing 2 mM Ca²⁺ and 2 mM Mg²⁺.When the cells are lysed by mechanical shearing the supernatant isdiluted with an equal volume of 2X Lysis buffer prior to mixing with ConA Sepharose. The supernatant is then allowed to bind to the Con ASepharose for 2-3 hours at 4° C. The material that fails to bind isharvested and dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for20 min. Then the resulting supernatant is harvested and applied to aMono Q FPLC column equilibrated in 20 mM Tris-Acetate pH 7.5, 20 mMNaCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. The column is thendeveloped with a 20 mM to 500 mM NaCl gradient and the eluted fractionsfractionated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and characterized by immunoblotting using anappropriate anti-Hsp70 antibody (such as from clone N27F3-4, fromStressGen).

[0045] Fractions strongly immunoreactive with the anti-Hsp70 antibodyare pooled and the Hsp70-peptide complexes precipitated with ammoniumsulfate; specifically with a 50%-70% ammonium sulfate cut. The resultingprecipitate is then harvested by centrifugation at 17,000 rpm (SS34Sorvall rotor) and washed with 70% ammonium sulfate. The washedprecipitate is then solubilized and any residual ammonium sulfateremoved by gel filtration on a Sephadex^(R) G25 column (Pharmacia).

[0046] The Hsp70-peptide complex can be purified to apparent homogeneityusing this method. Typically 1 mg of Hsp70-peptide complex can bepurified from 1 g of cells/tissue.

Purification of Hsp90-peptide Complexes

[0047] Initially, tumor cells are suspended in 3 volumes of 1X Lysisbuffer consisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2mM CaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF).Then, the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 min and then homogenized in a douncehomogenizer until >95% cells are lysed.

[0048] Then the lysate is centrifuged at 1000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of2X Lysis buffer prior to mixing with Con A Sepharose. The supernatant isthen allowed to bind to the Con A Sepharose for 2-3 hours at 4° C. Thematerial that fails to bind is harvested and dialyzed for 36 hours(three times, 100 volumes each time) against 10 mM Tris-Acetate pH 7.5,0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is centrifuged at17,000 rpm (Sorvall SS34 rotor) for 20 min. Then the resultingsupernatant is harvested and applied to a Mono Q FPLC columnequilibrated equilibrated with lysis buffer. The proteins are theneluted with a a salt gradient of 200 mM to 600 mM NaCl.

[0049] The eluted fractions are fractionated by SDS-PAGE and fractionscontaining the Hsp90-peptide complexes identified by immunoblottingusing a anti-Hsp90 antibody such as 3G3 (Affinity Bioreagents).Hsp90-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically, 150-200 μg of Hsp90-peptide complex can bepurified from 1 g of cells/tissue.

Purification of gp96-peptide Complexes

[0050] Initially, tumor cells are suspended in 3 volumes of 1X Lysisbuffer consisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2mM CaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF).Then, the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 min and then homogenized in a douncehomogenizer until >95% cells are lysed.

[0051] Then the lysate is centrifuged at 1000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and mixed with Con A Sepharose slurry equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺. When the cells are lysed bymechanical shearing the supernatant is diluted with an equal volume of2X Lysis buffer prior to mixing with Con A Sepharose. The supernatant isthen allowed to bind to the Con A Sepharose for 2-3 hours at 4° C. Theslurry is then packed into a column and washed with 1X lysis bufferuntil the OD₂₈₀ drops to baseline. Then the column is washed with ½column bed volume of 10% a-methyl mannoside (α-MM), the column sealedwith parafilm and incubated at 37° C. for 15 min. The column is thencooled to room temperature, the parafilm removed from the bottom of thecolumn, and five column volumes of a α-MM is applied to the column. Theeluate is then fractionated and characterized by SDS-PAGE. Typically,the resulting gp96-peptide complex is about 60 to 95% pure dependingupon the cell type and the tissue to lysis buffer ratio used.

[0052] If further purification is required, the sample can be applied toa Mono Q FPLC column equilibrated with a buffer containing 5 mM sodiumphosphate, pH7. The proteins are then eluted from the column with a 0-1MNaCl gradient. The gp96 fraction elutes between 400 mM and 550 mM NaCl.

[0053] As an alternative procedure, the gp96 fraction isolated from the100,000 g pellet can be resuspended in 5 volumes of PBS containing 1%sodium deoxycholate (without Ca²⁺ and Mg²⁺) and incubated on ice for 1h. The resulting suspension is centrifuged for 30 min at 20,000 g andthe resulting supernatant harvested and dialyzed against several changesof PBS (without Ca²⁺ and Mg²⁺) to remove the detergent. The resultingdialysate is centrifuged for 90 min at 100,000 g and the supernatantpurified further. Then calcium and magnesium are both added to thesupernatant to give final concentrations of 2 mM. Then the sample isapplied to a Mono Q HPLC column equilibrated with a buffer containing 5mM sodium phosphate, pH7 and the proteins eluted with a 0-1M NaClgradient. The gp96 fraction elutes between 400 mM and 550 mM NaCl.

[0054] The gp96-peptide complexes can be purified to apparenthomogeneity using this procedure. Typically about 10-20 μg of gp96 canbe isolated from 1 g cells/tissue using this method.

Formulation and Administration of the Complexes

[0055] Once stress protein-peptide complexes have been purified from theexcised tumor they are administered back to the mammal undergoingtherapy in order to stimulate in the mammal an immune response againsttumor cells from which the complex was derived. The stressprotein-peptide complexes of the invention may either be stored orprepared for administration by mixing with physiologically acceptablecarriers, excipients, or stabilizers. These materials should benon-toxic to the intended recipient at dosages and concentrationsemployed.

[0056] When the complex is water soluble it may be formulated in anappropriate buffer, for example PBS (5 mM sodium phosphate, 150 mM NaCl,pH7.1) or other physiologically compatible solutions. Alternatively, ifthe resulting complex has poor solubility in aqueous solvents then itmay be formulated with a non-ionic surfactant such as Tween, orpolyethylene glycol.

[0057] Useful solutions for oral or parenteral administration may beprepared by any of the methods well known in the pharmaceutical art,described, for example, in Remington's Pharmaceutical Sciences,(Gennaro, A., ed.), Mack Pub., 1990. Formulations may include, forexample, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes, and the like. Formulationsfor direct administration, in particular, may include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the stress protein-peptide complexes in vivo.

[0058] Formulations for inhalation may contain as excipients, forexample, lactose. Aqueous solutions may contain, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate. Oilysolutions may be useful administration in the form of nasal drops. Gelsmay be applied topically intranasally.

[0059] The compounds provided herein can be formulated intopharmaceutical compositions by admixture with pharmaceuticallyacceptable nontoxic excipients and carriers. In addition theformulations may optionally contain one or more adjuvants. Preferredadjuvants include, but are not limited to, pluronic tri-blockcopolymers, muramyl dipeptide and its derivatives, detoxified endotoxin,saponin and its derivatives such as QS-21 and liposomes. The presentinvention further envisages sustained release formulations in which thecomplex is released over an extended period of time.

[0060] The mode of administration of the family of stressprotein-peptide complexes prepared in accordance with the invention willnecessarily depend upon the stability of the complex under physiologicalconditions, and the size and distribution of the tumor within the mammalbeing treated. The preferred dosage of complex to be administered alsois likely to depend on such variables as the size and distribution ofthe tumor, the age, sex and weight of the intended recipient, theoverall health status of the particular recipient, the relativebiological efficacy of the complex, the formulation for the complex, thepresence and types of excipients in the formulation, and the route ofadministration.

[0061] In general terms, the compounds of this invention may be providedin an aqueous physiological buffer solution containing about 0.001 to10% w/v compound for parenteral administration. Preferred dosages rangefrom about 1 to about 1000 micrograms of complex/kg body weight ofrecipient/administration and most preferably range from about 100 toabout 250 micrograms of complex/kg body weight ofrecipient/administration. In particular, it is contemplated that atypical dose will range from about 5 mg to about 20 mg for a humansubject weighing about 75 kg. These quantities, however, may varyaccording to the adjuvant coadministered with the complex.

[0062] The complex preferably comprises part of an aqueous solutionwhich may be administered using standard procedures, for example,intravenously, subcutaneously, intramuscularly, intraorbitally,ophthalmically, intraventricularly, intracranially, intracapsularly,intraspinally, intracisternally, intraperitoneally, buccal, rectally,vaginally, intranasally or by aerosol administration. The aqueoussolution preferably is physiologically acceptable so that in addition todelivery of the desired complex to the mammal, the solution does nototherwise adversely affect the mammal's electrolyte and/or volumebalance. The aqueous medium for the complex thus may comprise normalphysiologic saline (0.9% NaCl, 0.15M), pH 7-7.4 or otherpharmaceutically acceptable salts thereof.

[0063] Preferably the recipient should be vaccinated three times at twoweek intervals. If necessary, the responses may be boosted at a laterdate by subsequent administration of the complex. It is contemplatedthat the optimal dosage and vaccination schedule may be determinedempirically for each stress protein-peptide complex using techniqueswell known in the art.

[0064] Various cytokines, antibiotics, and other bioactive agents alsomay be coadministered with the stress protein-peptide complexes. Forexample, various known cytokines, i.e., interleukin-1α (IL-1α),interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-3 (IL-3),interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6),interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9),interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12),interferon α (IFNα), interferon β (IFNβ), interferon γ (IFNγ), tumornecrosis factor α (TNF∝), tumor necrosis factor β (TNFβ), granulocytecolony stimulating factor (G-CSF), granulocyte/macrophage colonystimulating factor (GM-CSF), and transforming growth factor β (TGF-β)may be coadministered with the complexes in order to maximize thephysiological response. However, it is anticipated that other but as yetundiscovered cytokines may be effective in the invention. In addition,conventional antibiotics may be coadministered with the stressprotein-peptide complex. The choice of suitable antibiotics will howeverbe dependent upon the disease in question.

EXAMPLE I

[0065] In this example, C57BL/6 and C3H mice approximately 100 g inweight, are purchased from Jackson Laboratories, Bar Harbor, Me.Malignant tumor cells are then injected subcutaneously into mice inorder to induce experimental tumors in the mice. Specifically, malignantspindle cell carcinoma 6139 cells are injected subcutaneously into theC3H mice, malignant mouse Lewis lung carcinoma cells are injectedsubcutaneously into C57BL/6 mice and malignant mouse B16 melanoma cellsare injected subcutaneously into C57BL/6 mice.

[0066] When the tumors have grown to a size such that they are bothvisible and palpable, a sample of the tumor tissue is excised. As acontrol, normal non malignant tissue is excised from some mice bearingthe experimental tumors.

[0067] Then gp96-peptide, Hsp90-peptide and Hsp70-peptide complexes areisolated from both the excised normal and tumor derived tissues usingthe methods described hereinabove. Once isolated, the complexes arecombined with PBS and administered back to the mice from which thecomplexes were derived. Usually 6 mice are tested in each experiment.The experiments are performed using the schedule set forth below:

Experiment Composition administered back to mice

[0068] 1 gp96-peptide 2 Hsp70-peptide 3 Hsp90-peptide 4 gp96-peptide andHsp70-peptide 5 gp96-peptide and Hsp90-peptide 6 Hsp70-peptide andHsp90-peptide 7 Hsp70-peptide, Hsp90-peptide and gp96-peptide 8 bnfferalone

[0069] In one series of experiments the complexes are isolated fromtumor cells whereas in a second series the complexes are isolated fromnormal cells. The mice are inoculated three times at weekly intervalswith 20 micrograms (total weight) of the preselected complex(es). Duringtherapy, the size of each tumor is measured daily. After 4 weeks themice are sacrificed and the development of the tumor examinedhistologically. In addition, the sacrificed mice are examined for thepresence or absence of metastasis.

[0070] It is expected that the tumors in mice treated with complexesderived from normal tissue will continue to grow and metastasize. Incontrast, it is expected that the tumors in the mice treated with thecomplexes derived from the tumor tissue will be exhibit slower growththan the tumors in the control animals, and in some cases, it isexpected that the tumor mass may get smaller and the tumor exhibitremission during therapy.

Other Embodiments

[0071] The invention may be embodied in other specific forms withoutdeparting from the spirit or essential characteristics thereof. Thepresent embodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

What is claimed is:
 1. A method for inhibiting proliferation of a tumorin a mammal, the method comprising: administering to the mammalharboring the tumor a composition comprising, (a) an immunogenic stressprotein-peptide complex isolated from a cell derived from the tumor,said complex being operative to initiate in the mammal an immuneresponse against said tumor, and (b) a pharmaceutically acceptablecarrier, in an amount sufficient to elicit in the mammal an immuneresponse against the tumor thereby inhibiting proliferation of thetumor.
 2. The method of claim 1, wherein the stress protein in thecomplex is a Hsp70, a Hsp90 or a gp96.
 3. The method of claim 1, whereina peptide in the complex is non covalently associated with the stressprotein.
 4. The method of claim 1, wherein administering the complexinitiates an immune response mediated by a T cell.
 5. The method ofclaim 4, wherein administering the complex initiates an immune responsemediated by a cytotoxic T cell.
 6. The method of claim 1, wherein thecomplex is administered to the mammal in an amount in the range of about1 to about 1000 micrograms of complex/kg body weight ofmammal/administration.
 7. The method of claim 6, wherein said amount isin the range of about 100 to about 250 micrograms of complex/kg bodyweight of mammal/administration.
 8. The method of claim 1, wherein thecomplex is administered repeatedly to the mammal.
 9. The method of claim1, wherein the composition is administered to the mammal in combinationwith a cytokine.
 10. A method for inhibiting proliferation of a tumor ina mammal, the method comprising the steps of: (a) providing a tumor cellexcised from the mammal, (b) isolating from the cell an immunogenicstress protein-peptide complex operative to initiate in the mammal animmune response against the tumor cell, and (c) administering to themammal the isolated stress protein-peptide complex in an amountsufficient to elicit in the mammal an immune response against the tumorcell thereby to inhibit proliferation of any tumor cell remaining in themammal.
 11. The method of claim 10, wherein the stress protein in thecomplex is a Hsp70, a Hsp90 or a gp96.
 12. The method of claim 10,wherein a peptide in the complex is non covalently associated with thestress protein.
 13. The method of claim 10, wherein administering thecomplex initiates an immune response mediated by a T cell.
 14. Themethod of claim 13, wherein administering the complex initiates animmune response mediated by a cytotoxic T cell.
 15. The method of claim10, wherein the complex is administered to the mammal in an amount inthe range of about 1 to about 1000 micrograms of complex/kg body weightof mammal/administration.
 16. The method of claim 15, wherein saidamount is in the range of about 100 to about 250 micrograms ofcomplex/kg body weight of mammal/administration.
 17. The method of claim10, wherein the complex is administered repeatedly to the mammal. 18.The method of claim 10, wherein said complex is administered to themammal in combination with a cytokine.